Cardiac myocytes constitutively express enzymes termed neuronal nitric oxide synthase (nNOS, NOS1) and endothelial nitric oxide synthase (eNOS, NOS3). Nitric oxide (NO) produced via NOS1 and NOS3 is an important regulator of cardiac contractility in the normal, healthy heart. NOS1 and NOS3 also play a key role in the remodeling and cardiac dysfunction of diseased hearts. NOS1 has been found to be essential for a positive force-frequency response and to potentiate the beta-adrenergic response. This is controversial, as other studies have found somewhat dissimilar results. The effects of NOS3 are better understood, in that NOS3 limits the beta-adrenergic response. However, definitive mechanisms, end targets and signaling pathway(s) for NOS1 and NOS3 are not known. Thus, the objective of this proposal is to critically examine how NOS1 and NOS3 regulate cardiac myocyte function. The proposed experiments, using state-of-the-art electrophysiology and optical methods, as well as biochemical assays, will evaluate the effects of NOS1 and NOS3 on cardiac myocyte function. The results of these studies will provide a new integrated understanding of how NOS1 and NOS3 regulate cardiac myocyte function and will help delineate their role in cardiac disease states. The specific aims are designed to examine the mechanisms, end targets and signaling pathways of NOS1 and NOS3. The hypothesis is that NOS1 and NOS3 differentially regulate cardiac contractility by ultimately modulating sarcoplasmic reticulum Ca2+ load through distinct end targets and signaling pathways. The specific aims are to: 1) define physiological effects of NOS1 and NOS3 on cardiac myocyte function;2) determine mechanisms of NOS1 and NOS3 by examining specific end targets;3) elucidate the downstream pathways of NOS1 or NOS3 on sarcoplasmic reticulum Ca2+ ATPase, phospholamban and the L-type Ca2+ channel. Studies will be performed on myocytes and trabeculae isolated from wildtype, NOS1 knockout, NOS3 knockout and phospholamban knockout mice, as well as rabbits. Heart disease is one of the leading causes of mortality in the Western world. The relevance of this proposal to public health is that these results may improve the treatment of heart disease by potentially identifying new targets for drug development.